Polystyrene nanoparticles (PS NPs) as proxy for nanoplastics for studying uptake, biodisposition, morphological and biological effects on Ciona robusta embryos, larval, juvenile and adult stages.

Plastic pollution in marine environments is one of the most pressing ecological challenges of the 21st century. Over 14 million tons of plastic waste enter the oceans annually, and as plastic debris fragments into smaller particles, including microplastics and nanoplastics (<1µm), it has become a significant concern. Due to their small size and high dispersal capacity, nanoplastics can accumulate in surface waters, where they may interact with planktonic species and early life stages of benthic invertebrates, enhancing their bioavailability. However, the ecological risks posed by nanoplastics—particularly in terms of bioaccumulation and biological responses—remain poorly understood. Synthetic polystyrene nanoparticles (PS NPs), which are most commonly used as proxies for nanoplastics, have been widely employed in ecotoxicological studies. These particles, depending on their surface charge and functionalization, can cause developmental and immune toxicity, particularly during the early life stages of marine invertebrates. The embryotoxic effects of PS NPs have been well-documented across various marine species, including fish, crustaceans, mollusks, and more recently, ascidian species like Ciona robusta. The transparency and simplicity of C. robusta during early developmental stages, combined with its rapid life cycle, fully sequenced genome, and ease of laboratory culture, make it an ideal model organism for studies in developmental biology, evolutionary research, and toxicological effects of environmental contaminants like nanoplastics. Furthermore, C. robusta is an ecologically significant marine invertebrate, playing a vital role in marine ecosystems as a filter feeder from early developmental stages. This behavior increases its exposure to plastics and associated contaminants, making it a relevant species for studying the impact of environmental pollutants. Exposure to nanoplastics can interfere with cellular processes, gene expression, and developmental mechanisms, leading to malformations, growth delays, and reduced survival. Nanoplastics do not act in isolation in marine ecosystems; their uptake and ecotoxicity may be influenced by the presence of other environmental pollutants. Among emerging contaminants, plastic additives, particularly Bisphenol A (BPA), have attracted significant attention. These chemicals, often leached into marine environments alongside plastics, can adsorb to the surface of nanoplastics, altering their bioavailability and enhancing their toxicity. BPA, a widely used industrial chemical in plastic production, is a well-known endocrine disruptor with a broad range of biological effects. It interferes with reproductive development and endocrine system function in both aquatic and terrestrial organisms. Its widespread presence in marine environments, combined with its potential for bioaccumulation, raises significant concerns about its effects on marine species, particularly during the vulnerable early developmental stages. The interaction between BPA and nanoplastics could magnify the adverse effects on marine organisms, highlighting the importance of studying their combined impacts. While significant research has been conducted on the individual effects of nanoplastics and BPA, a critical gap remains in understanding their combined effects. Studies exploring the co-exposure of nanoplastics and emerging contaminants like BPA are essential for fully assessing ecological risks. Additionally, while the biological impacts of nanoplastics have been widely studied, significant challenges remain in tracking their localization and accumulation within biological matrices. Radiolabeling NPs with isotopes, such as 14C, provides a promising method for investigating their biodistribution and bioaccumulation in organisms. This approach enhances the sensitivity and accuracy of in vivo tracking, enabling the observation of how NPs accumulate in various tissues over time. However, radiolabeling requires careful optimization to ensure stable and efficient labeling while maintaining the biological relevance of the nanoparticles. This thesis investigates the exposure of C. robusta to nanoplastics using PS NPs and BPA, both alone and in combination, during larval and juvenile stages. It further explores the uptake and potential transgenerational impacts in adults exposed to radiolabeled 14C PS NPs. Through in vitro and in vivo experiments, the study evaluates the acute and chronic effects of PS NPs and BPA, emphasizing gene modulation linked to oxidative stress, detoxification mechanisms, and potential neurotoxicity by analyzing acetylcholinesterase (AChE) activities. The results indicate that BPA exposure in vitro inhibited AChE activity, while PS NPs did not affect this enzyme. Both contaminants, alone and combined, triggered significant changes in the expression of genes associated with oxidative stress and detoxification. Chronic exposure to PS NPs resulted in morphological alterations in juveniles and increased oxidative stress. Additionally, exposure to 14C-labeled PS NPs led to their accumulation in adult internal organs, suggesting potential transgenerational effects. These findings enhance our understanding of the combined impact of PS NPs and BPA on marine organisms, underscoring broader implications for marine ecosystem health and the management of emerging contaminants. This research provides new insights into the adaptive and resistance mechanisms of marine invertebrates, addressing critical gaps in knowledge regarding the effects of nanoplastics and chemical pollutants on marine life.